Gram-negative bacterial diseases are important causes of morbidity and mortality throughout the world. Our research focuses on the role of gram-negative bacterial lipopolysaccharides (LPS) in pathogenesis. In this application for continuation of an existing grant, we propose to explore in detail the metabolism of LPS by human and mouse cells. Lipid A, the toxic moiety of the LPS, is a glucosamine disaccharide to which fatty acids, phosphates, and the polysaccharide chain are covalently attached. Hydroxylated fatty acids (usually 3-hydroxytetradecanoate) are linked directly to glucosamine, whereas normal fatty acids (principally dodecanoic and tetradecanoic acids) are attached to glucosamine by being substituted to the hydroxyl group of a glucosamine-linked hydroxylated fatty acid. We have recently discovered that human neutrophils contain enzymes that specifically deacylate lipid A by hydrolysing the acyloxyacyl linkages, thus releasing the normal fatty acids. Since glucosamine-linked fatty acids (particularly the hydroxylated fatty acids) are thought to be necessary for LPS to have biological activity, we would like to determine the effects of this enzymatic deacylation on the activity of the LPS. The proposed research will investigate (1) the biological properties of partially deacylated LPS (2) the effects of compounds that inhibit lipid A deacylation, (3) quantitative and qualitative aspects of lipid A degradation in human peripheral blood neutrophils and monocytes, and (4) lipid A metabolism by macrophages from normal mice and from mice that have a genetic defect that renders them hyporesponsive to the toxicity of LPS. These studies should provide important new information about the mechanism(s) by which LPS stimulate cells and cause toxicity in animals, as well as new insights concerning the host mechanism(s) for inactivating these potent molecules. We hope that they will improve the scientific basis for advances in the therapy of diseases caused by gram-negative bacteria.